Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the unicellular green alga Chlamydomonas reinhardtii, phototropin (PHOT) plays a vital role in the progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, and chlorophyll binding proteins. The UV-A/blue light sensing phototropins mediate a variety of light responses and are responsible in higher plants for optimization of photosynthetic yields (Chen, Chory et al. 2004).
Phototropins are commonly composed of two domains, an amine terminal photosensory domain and a carboxy terminal serine/threonine protein kinase domain. The photosensory domain is a flavin mononucleotide binding domain, the LOV domain. Plants and green algae contain two of these domains in the phototropin regulatory sequence, LOV1 and LOV2 (Chen, Chory et al. 2004).
Phototropin knock-out mutants (PHOT K/O) have been made previously in plants (Suetsugu and Wada 2007, Moni, Lee et al. 2015) and algae (Zorin, Lu et al. 2009; Trippens, Greiner et al. 2012). However, all the PHOT K/O mutant prior art that has been located to date did not show improved productivity of the plant or alga.
In plants two phototropins have been reported, phot1 and phot2, these phototropins share sequence homology and have overlapping functions. These blue-light-sensitive receptors consist of two parts: a C-terminal serine-threonine kinase and two LOV domains that bind flavin mononucleotide as chromophores at the N-terminus. Recently, in the unicellular green alga, Chlamydomonas reinhardtii, a phototropin homolog was identified. It exhibits photochemical properties similar to those of higher plant phototropins and is also functional in Arabidopsis. Studies show that the basic mechanism of phototropin action is highly conserved, even though its apparent physiological functions are quite diverse.
Phototropin in Higher Plants:
Plants utilize several families of photoreceptors to better react to their environment, allowing them to fine tune pathways controlled by the photoreceptors—phototropin, phytochrome, and cryptochrome (Chen, Chory et al. 2004).
In higher plants phototropin mediates a variety of blue-light elicited physiological processes (Sullivan, Thomson et al. 2008). Phototropins are UV-A/blue light sensing photoreceptors that are known to optimize photosynthetic yields (Chen, Chory et al. 2004). The involvement of phototropin in photomovement in higher plants is well documented (Suetsugu and Wada 2007, Kagawa, Kimura et al. 2009). Studies involving Arabidopsis mutants lacking the phot1 and phot2 genes have revealed that in addition to regulating hypocotyl curvature of seedlings towards blue light, phototropins also regulate a diverse range of responses in flowering plants. These responses include chloroplast movements, nuclear positioning, stomatal opening, leaf expansion, leaf movements and leaf photomorphogenesis.
Phototropin knock-out mutants (PHOT K/O) have been made previously in plants (Suetsugu and Wada 2007, Moni, Lee et al. 2015). For instance in Physcomitrella patens (a moss) there are three PHOT genes and they have all been knocked out in different mutants (Suetsugu and Wada 2007). The focus of the P. patens study was the effect of PHOT K/O on phototropism (movement toward light) and the phenotypes they observed allowed them to determine which of the genes were necessary for phototropism (Suetsugu and Wada 2007).
PHOT expression was higher in darkness than in light, and phot1 Arabidopsis mutants was shown to increase the number of lateral roots produced (Moni, Lee et al. 2015). phot was also demonstrated to mediate phototropism, chloroplast relocation and leaf expansion (Matsuoka, Iwata et al. 2007). Using phot deficient Arabidopsis mutants, phototropin 2 was linked to palisade parenchyma cell development of leaves (Kozuka, Kong et al. 2011).
Another study looked at the role of phototropin under low photosynthetically active radiation (Takemiya, Inoue et al. 2005). They found that the wild-type and the PHOT1 mutant both showed increased but similar growth in low radiance blue light super imposed on red light. In white light there was no increase in biomass in both phot1 and phot2 mutants as well as in the double phot mutant.
A study by Folta and colleagues investigated the relationship between phot1 and phototropism and growth inhibition in Arabidopsis (Folta, Lieg et al. 2003). They found that the onset of phototropism and the phot1-mediated growth inhibition coincided and postulated that both were due to phot1 expression.
There is a substantial amount of patent literature around phototropin in higher plants. However, the focus has been on the commercial utility of the upstream, light regulated areas rather than on the phototropin gene itself. These light control domains that regulate PHOT expression—the light-oxygen-voltage-sensing (LOV) domains—have been carefully evaluated for potential commercial application in higher plants.
Shu & Tsien application (US20130330718) focused on using the LOV domain for control of proteins that generate singlet oxygen (SOGs). These fusion protein tags could be used for imaging under blue light for research purposes.
Other patents use light switchable regulatory sequences and contemplate the use of the phototropin LOV domain such as Yang and colleagues (EP2682469).
Hahn & Karginov (WO2011133493) focused on allosteric regulation of kinases using the light activated domains for control of expression in engineered fusion proteins (such as the LOV domains).
Hahn and colleagues (U.S. Pat. No. 8,859,232) demonstrated that the LOV domain of phototropin can be used as a light activated switch for the activation or inactivation of fusion proteins of interest. They contemplated using a LOV domain that could contain substantial portions of the phototropin molecule in addition to the LOV domain. They contemplated using the LOV domain isolated from algae and gave the specific example of Vaucheria frigida, a stramenopile or heterokont alga.
Kinoshita and colleagues (WO2014142334) demonstrated that overexpression of phototropin had no impact of stomatal opening in higher plants.
Bonger and colleagues (US20140249295) used the LOV domain as a fusion with another functional protein wherein the light switching ability of the LOV domain was used to control the stability and/or function of the fusion protein.
Folta and colleagues (WO2014085626) using mutants of phototropin 1 were able to show that the function of phot1 is mediation of the pathway in which green light reverses the effects of red and/or blue light on plant growth.
Schmidt & Boyden (US20130116165) describe a new group of fusion proteins with light regulatory regions derived from Avena sativa phototropin 1. These regulatory domains are used for altering channel function in membranes.
To date there is no disclosure of the use of PHOT knockout or knockdown (suppression) technology to improve or algae plant productivity.
Phototropin in Algae:
Phototropin has already been well studied in several different algae including Chlamydomonas reinhardtii (Briggs and Olney 2001). However, there are indications that phototropins have diverged significantly or that the genes that function as phototropin are not very homologous to plant phototropin genes. For instance it was reported that in Thalassiosira pseudonana (a diatom) and Cyanidioschyzon merolae (unicellular red alga) no genes were found encoding the phototropins (Grossman 2005). However putative genes with photosensory LOV domains, aurechromes, have been reported for these and other photosynthetic stramenopiles (Table 1). Most aureochromes contain a single LOV domain and function as transcription factors that regulate cell division, chloroplast movement, pigment production, and phototropism. (Takahashi. J Plant Res (2016) 129:189-197)
In Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle (Huang and Beck 2003), control of the eye spot size and light sensitivity (Trippens, Greiner et al. 2012). Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. Phototropin has been localized to the flagella of Chlamydomonas reinhardtii (Huang, Kunkel et al. 2004). Phototropin is also known to be involved in expression of genes encoding chlorophyll and carotenoid biosynthesis and LHC apoproteins in Chlamydomonas reinhardtii (Im, Eberhard et al. 2006). The Chlamydomonas reinhardtii phototropin gene has been cloned and shown to function when expressed in Arabidopsis (Onodera, Kong et al. 2005).
Phototropin has been shown to control multiple steps in the sexual life cycle of Chlamydomonas reinhardtii (Huang and Beck 2003). PHOT knockdowns using RNAi were generated (Huang and Beck 2003). The entire focus of this study was on sexual mating and no mention of improved biomass, starch accumulation or photosynthesis rate was observed. It is also involved in the chemotaxis that is the initial phase of the sexual cycle of Chlamydomonas reinhardtii (Ermilova, Zalutskaya et al. 2004). However, no cell cycle implications of phototropin knockout or knockdowns have been published.
Detailed studies have carefully analyzed the function of the LOV domain in several algal species. An example is the Chlamydomonas reinhardtii mutant LOV2-C250S where careful studies of the light activation and regulation of this domain were carried out to better understand the mechanism of action (Sethi, Prasad et al. 2009).
Phototropin knock-out mutants (PHOT K/O) have been made previously in algae (Zorin, Lu et al. 2009 Trippens, Greiner et al. 2012). PHOT minus strains had larger eyespots than the parental strain (Trippens, Greiner et al. 2012). This study focused on the impact of PHOT on eyespot structure function. These authors used a knock-out mutant of PHOT to reduce expression of phototropin (Trippens, Greiner et al. 2012).
Novel phototropins have been described in the green alga Ostreococcus tauri and with a focus on their LOV domain structure/function (Veetil, Mittal et al. 2011).
Abad and colleagues (WO2013056212) provide the sequence for phototropin from a green alga, Auxenochlorella protothecoides, and indicate that the gene would be important for photosynthetic efficiency. However, they do not discuss the impact of deletion or inhibition of this gene on the alga.